The molecular mechanisms linking environmental and genetic factors in obesity-induced cardiac diseases remain poorly understood. We previously identified a metabolic-responsive super-enhancer (SE) in the mouse (C57BL6J) heart following high-fat diet (HFD) exposure. To dissect its functional role, we generated cardiomyocyte-specific metabolic enhancer knockout (cME-KO) lines targeting distinct SE regions using CRISPR/Cas9. Loss of the SE attenuated the HFD-driven induction of fatty acid metabolism (FAM) genes, including Pdk1, Acot3, Cpt1b, and Gpam, indicating that this SE governs diet-induced metabolic reprogramming in cardiomyocytes (e.g., Pdk1 fc WT 1.21 vs. ME-cKO 1.05, Acot3 fc WT 2.39 ME-cKO 2.19, Cpt1b fc WT 1.28 vs. ME-cKO 1,09 and Gpam fc WT 1.52 vs. ME-cKO 1.29.
To characterize transcriptional regulators associated with the cardiac SE, we performed a biotin-labeled SE pulldown coupled to mass spectrometry. This revealed canonical SE-associated cofactors such as Med1 and Brd4, as well as retinoid X receptors (RXRs) as key transcriptional mediators.
We hypothesized, that aggregation of positively charged proteins would be supported by small negatively charged RNAs, such as enhancer RNAs (eRNA). We identified an intrinsically disordered region (IDR) within RXR-α (amino acids 169–189) in the DisProt database. These regions (IDRs) are known to phase separate under distinct conditions to form complexes with transcriptional activity. A recombinant GFP-RXR-α IDR was the incubated with purified SE-derived small noncoding RNAs (eRNAs) in droplet buffer (pH 7.4). The RXR-α IDR underwent phase separation when incubated with eRNAs in a concentration-dependent manner, forming more pronounced condensates in control of scrambled RNA (fold-change droplet size: 1.75; fold-change normalized fluorescence intensity: 3.18; p < 0.001, Mann–Whitney U test). In mammalian cells, co-expression of the eRNA and RXRα-GFP promoted nuclear condensate formation, consistent with enhancer-associated phase separation and transcriptional hotspot activity.
In conclusion, our data suggest a novel mechanism of energy independent transcriptional regulation in the obese heart. This is controlled by eRNA-dependent nuclear phase separation of the retinoid acid receptor.
